Novel glycosyltransferases and polynucleotides encoding the same

ABSTRACT

The present invention provides novel glycosyltransferase proteins produced by ascomycetous filamentous fungi (preferably species belonging to the genus  Trichoderma,  more preferably  Trichoderma viride ), as well as genes encoding the same. Among novel enzyme proteins provided by the present invention, particularly preferred is an enzyme protein obtained from the culture supernatant of  Trichoderma viride  strain IAM5141. The novel enzymes of the present invention allow glycosylation of flavonoid compounds to thereby improve their water solubility.

RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.12/523,068, which is a National Stage Application of InternationalApplication No. PCT/JP2008/050618, filed Jan. 18, 2008, which claimspriority to Japanese Application No. 2007-010759, filed Jan. 19, 2007.The entire disclosures of U.S. application Ser. No. 12/523,068 andInternational Application No. PCT/JP2008/050618 are considered as beingpart of this application and are expressly incorporated by referenceherein in their entireties.

TECHNICAL FIELD

The present invention relates to novel glycosyltransferases which allowglycosylation of flavonoid compounds, and polynucleotides encoding thesame. Glycosides of flavonoid compounds obtained by the presentinvention can be used for food, pharmaceutical and cosmetic purposes.

BACKGROUND ART

Proanthocyanidin (grape seed extract) has been studied for itsusefulness as a therapeutic agent for blood vessels, and one of thereasons for recent progress in these studies is that the targetsubstance can serve as a marker for tracing in vivo absorption andmetabolism because it is stable against heat and acids, highly solublein water and highly absorbable in the body. In contrast, polyphenolcompounds such as catechin are often difficult to dissolve in water, andalso involve a problem in that they are less absorbable in the body.

Attempts have been made to develop a technique for glycosylation ofcatechin and other compounds, with the aim of improving their solubilityin water and increasing their stability.

By way of example, Patent Document 1 discloses a-glucosidase with amolecular weight of about 57,000, which was collected from a culturesolution of Xanthomonas campestris WU-9701. This enzyme uses maltose orthe like as a donor (does not use maltotriose, cyclodextrin or starch asa donor) and transfers glucose to a specific acceptor to synthesize aglycoside. In this document, compounds listed as acceptors are thosehaving an alcoholic hydroxyl group (e.g., menthol, ethanol, 1-propanol,1-butanol, 2-butanol, isobutyl alcohol, 1-amyl alcohol, 2-amyl alcohol,5-nonyl alcohol), as well as those having a phenolic hydroxyl group(e.g., capsaicin, dihydrocapsaicin, nonylic acid vanillylamide,catechin, epicatechin, vanillin, hydroquinone, catechol, resorcinol,3,4-dimethoxyphenol). Moreover, glycosides whose production was actuallyconfirmed are monoglucosides only.

Patent Document 2 discloses a method in which a mixture of a catechincompound and glucose-l-phosphate or sucrose is treated with sucrosephosphorylase to prepare a glycoside of the catechin compound. Thesources of sucrose phosphorylase listed therein are Leuconostocmesenteroides, Pseudomonas saccharophila, Pseudomonas putrefaciens,Clostridium pasteurianum, Acetobacter xylinum, and Pullularia pullulans.Likewise, catechin compounds listed as acceptors are (+)-catechin,(−)-epicatechin, (−)-epicatechin 3-O-gallate, (−)-epigallocatechin and(−)-epigallocatechin 3-O-gallate, but it is only (+)-catechin that wasactually used as an acceptor to prepare (+)-catechin3′-O-α-D-glucopyranoside in the Example section.

Patent Document 3 discloses epigallocatechin 3-O-gallate derivatives, inwhich a glucose residue or a maltooligosaccharide residue with apolymerization degree of 2 to 8 is attached to at least one of the 5-,7-, 3′-, 4′-, 5′-, 3″-, 4″- and 5″-positions. As in the case of PatentDocument 2, the Example section of Patent Document 3 actually disclosesonly a case where a mixture of (−)-epigallocatechin gallate andglucose-l-phosphate or sucrose was treated with sucrose phosphorylase toprepare 4′-O-α-D-glucopyranosyl(−)-epigallocatechin gallate and4′,4″-O-α-D-di-glucopyranosyl(−)-epigallocatechin gallate.

Patent Document 4 discloses tea extracts or tea beverages whoseastringent taste is reduced by glycosylation of polyphenols containedtherein. To reduce the astringent taste of tea extracts or teabeverages, this document describes detailed procedures in which teaextracts or tea beverages are supplemented with dextrin, cyclodextrin,starch or a mixture thereof, and then treated with cyclomaltodextringlucanotransferase. In the Example section, it is shown that a green teaextract and α-cyclodextrin were treated with cyclomaltodextringlucanotransferase derived from Bacillus stearothermophilus to give areaction product with reduced astringent taste, which in turn indicatesthat polyphenols such as epigallocatechin 3-O-gallate and epicatechinwere glycosylated. However, this document fails to show the detailedstructure of the reaction product.

Patent Document 5 discloses glycosides of catechin compounds in whichglycosylation occurs at the 3′-position, at the 3′- and 5-positions, orat the 3′- and 7-positions. For this purpose, this document describesdetailed procedures in which a catechin compound and dextrin,cyclodextrin, starch or a mixture thereof are treated withcyclomaltodextrin glucanotransferase derived from Bacillusstearothermophilus, as in the case of Patent Document 4. Further, in theexamples using dextrin as a glycosyl donor in the above procedures, someof the resulting glycosides of (−)-epigallocatechin,(−)-epigallocatechin 3-O-gallate and (−)-epicatechin 3-O-gallate areconsidered to have 6 to 8 glucose residues on average per molecule ofeach polyphenol, as determined from their molar absorption coefficients.Moreover, it is confirmed that upon treatment with glucoamylase derivedfrom Rhizopus niveus, the glycosides obtained by the above proceduresgenerated 3′,7-di-O-α-D-glucopyranosyl(−)-epigallocatechin,3′,5-di-O-α-D-glucopyranosyl(−)-epigallocatechin,3′-O-α-D-glucopyranosyl(−)-epigallocatechin,3′,7-di-O-α-D-glucopyranosyl(−)-epigallocatechin 3-O-gallate,3′-O-α-D-glucopyranosyl(−)-epigallocatechin 3-O-gallate,3′-O-α-D-glucopyranosyl(−)-gallocatechin and3′-O-α-D-glucopyranosyl(−)-epicatechin 3-O-gallate.

As to effects provided by catechin glycosides, Non-patent Document 1describes reduced astringent taste, increased water-solubility, improvedstability and inhibited tyrosinase, while Non-patent Document 2describes suppressed mutagenicity.

-   -   Patent Document 1: JP 2001-46096 A    -   Patent Document 2: JP 05-176786 A (Japanese Patent No. 3024848)    -   Patent Document 3: JP 07-10897 A (Japanese Patent Patent        Document 5: JP 09-3089 A (Japanese Patent No. 3712285)    -   Non-patent Document 1: Biosci. Biotech. Biochem., 57 (10),        1666-1669 (1993)    -   Non-patent Document 2: Biosci. Biotech. Biochem., 57 (10),        1290-1293 (1993)

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

These glycosylation techniques cannot be regarded as sufficient in termsof properties of the enzymes used therein, including glycosyl donorspecificity, specificity to compounds which can be glycosylated,glycosylation efficiency, etc. Thus, there has been a demand for thedevelopment of glycosyltransferases with more excellent properties.

Means for Solving the Problems

The inventors of the present invention have made extensive and intensiveefforts to develop a glycosylation technique for flavonoid includingcatechin. As a result, the inventors have found a novelglycosyltransferase from the culture supernatant of Trichoderma virideand have cloned a gene thereof, thereby completing the presentinvention.

[Glycosyltransferase genes and others]

The present invention provides a polynucleotide or a homolog thereof,which encodes a glycosyltransferase protein produced by ascomycetousfilamentous fungi (preferably species belonging to the genusTrichoderma, more preferably Trichoderma viride), i.e., a polynucleotidecomprising any one of (A) to (K) shown below (preferably apolynucleotide consisting of any one of (A) to (K) shown below):

(A) a polynucleotide which consists of all or part of the nucleotidesequence shown in SEQ ID NO: 6 (e.g., all or part of nucleotides423-1928, a region corresponding to the nucleotide sequence of SEQ IDNO: 9);

(B) a polynucleotide which is hybridizable under stringent conditionswith a polynucleotide consisting of a nucleotide sequence complementaryto the nucleotide sequence of the polynucleotide shown in (A) and whichencodes a protein having glycosylation activity on a flavonoid compound;

(C) a polynucleotide which consists of a nucleotide sequence comprisingsubstitution, deletion, insertion and/or addition of one or severalnucleotides in the nucleotide sequence of the polynucleotide shown in(A) and which encodes a protein having glycosylation activity on aflavonoid compound;

(D) a polynucleotide which shares an identity of at least 60% or morewith the nucleotide sequence of the polynucleotide shown in (A) andwhich encodes a protein having glycosylation activity on a flavonoidcompound;

(E) a polynucleotide which consists of the nucleotide sequence shown inSEQ ID NO: 9;

(F) a polynucleotide which is hybridizable under stringent conditionswith a polynucleotide consisting of a nucleotide sequence complementaryto the nucleotide sequence of the polynucleotide shown in (E) and whichencodes a protein having glycosylation activity on a flavonoid compound;

(G) a polynucleotide which consists of a nucleotide sequence comprisingsubstitution, deletion, insertion and/or addition of one or severalnucleotides in the nucleotide sequence of the polynucleotide shown in(E) and which encodes a protein having glycosylation activity on aflavonoid compound;

(H) a polynucleotide which shares an identity of at least 60% or morewith the nucleotide sequence of the polynucleotide shown in (E) andwhich encodes a protein having glycosylation activity on a flavonoidcompound;

(I) a polynucleotide which encodes a protein consisting of the aminoacid sequence shown in SEQ ID NO: 10;

(J) a polynucleotide which encodes a protein consisting of an amino acidsequence comprising substitution, deletion, insertion and/or addition ofone or several amino acids in the amino acid sequence shown in SEQ IDNO: 10 and having glycosylation activity on a flavonoid compound; and

(K) a polynucleotide which encodes a protein consisting of an amino acidsequence sharing an identity of at least 60% or more with the amino acidsequence shown in SEQ ID NO: 10 and having glycosylation activity on aflavonoid compound.

SEQ ID NO: 6 shows the nucleotide sequence of genomic DNA forglycosyltransferase TRa2 obtained from T. viride strain IAM5141, whileSEQ ID NOs: 9 and 10 show the cDNA and deduced amino acid sequencesthereof, respectively.

When the deduced amino acid sequence of TRa2 is analyzed by Signal P, asequence covering amino acids 1-20 of SEQ ID NO: 10 is predicted as asecretion signal. Thus, a mature protein essential for serving as aglycosyltransferase lies in the sequence downstream of amino acid 21 inSEQ ID NO: 10; it would be suitable to remove the signal sequence ifthis enzyme is expressed in a heterologous expression system such as E.coli. Polynucleotides encoding such a mature protein also fall withinthe scope of the present invention.

Thus, the present invention also provides a polynucleotide comprisingany one of (L) to (R) shown below (preferably a polynucleotideconsisting of any one of (L) to (R) shown below):

(L) a polynucleotide which consists of the nucleotide sequence shown inSEQ ID NO: 25;

(M) a polynucleotide which is hybridizable under stringent conditionswith a polynucleotide consisting of a nucleotide sequence complementaryto the nucleotide sequence shown in SEQ ID NO: 25 and which encodes aprotein having glycosylation activity on a flavonoid compound;

(N) a polynucleotide which consists of a nucleotide sequence comprisingsubstitution, deletion, insertion and/or addition of one or severalnucleotides in the nucleotide sequence of the polynucleotide shown inSEQ ID NO: 25 and which encodes a protein having glycosylation activityon a flavonoid compound;

(O) a polynucleotide which shares an identity of at least 60% or morewith the nucleotide sequence shown in SEQ ID NO: 25 and which encodes aprotein having glycosylation activity on a flavonoid compound;

(P) a polynucleotide which encodes a protein consisting of the aminoacid sequence shown in SEQ ID NO: 26;

(Q) a polynucleotide which encodes a protein consisting of an amino acidsequence comprising substitution, deletion, insertion and/or addition ofone or several amino acids in the amino acid sequence shown in SEQ IDNO: 26 and having glycosylation activity on a flavonoid compound; and

(R) a polynucleotide which encodes a protein consisting of an amino acidsequence sharing an identity of at least 60% or more with the amino acidsequence shown in SEQ ID NO: 26 and having glycosylation activity on aflavonoid compound.

SEQ ID NO: 25 shows the nucleotide sequence of cDNA encoding TRa2 as amature protein, i.e., a nucleotide sequence covering nucleotides 61-1389of SEQ ID NO: 9. Likewise, SEQ ID NO: 26 shows the amino acid sequenceof TRa2 as a mature protein, i.e., an amino acid sequence covering aminoacids 21-463 of SEQ ID NO: 10.

The present invention also provides a polynucleotide which is derivedfrom the genus Trichoderma (preferably Trichoderma viride, Trichodermareesei, Trichoderma saturnisporum, Trichoderma ghanense, Trichodermakoningii, Trichoderma hamatum, Trichoderma harzianum or Trichodermapolysporum), comprises any one of the nucleotide sequences shown in SEQID NOs: 11 to 24, and encodes a protein having glycosylation activity ona flavonoid compound. A preferred example of such a polynucleotide is apolynucleotide which comprises any one of the nucleotide sequences shownin SEQ ID NOs: 11 to 24, shares high identity with the nucleotidesequence shown in SEQ ID NO: 6, 9 or 25, and encodes a protein havingglycosylation activity on a flavonoid compound.

As used herein, the term “glycosylation activity” is intended to meanhaving the ability to transfer a sugar residue to a flavonoid compound.To confirm whether a protein has the ability to transfer a sugar residueto a flavonoid compound, unless otherwise specified, a mixture offlavonoid (e.g., catechin) and an appropriate glycosyl donor (e.g.,dextrin) may be contacted with the target enzyme and reacted for asufficient period of time, followed by analysis of the reaction solutionthrough high performance liquid chromatography (HPLC) or othertechniques, for example as shown in the Example section described later.

A protein encoded by the polynucleotide of the present invention mayhave not only glycosylation activity, but also an additional activity,such as dextrinase activity. As used herein, the term “dextrinase” isintended to mean an enzyme capable of hydrolyzing carbohydratescontaining α-glucoside linkages (e.g., starch, dextrin), unlessotherwise specified. Dextrinase is a kind of amylase. To determinewhether a target has dextrinase activity, a commercially availabledextrin (e.g., starch hydrolyzed with an acid, heat or an enzyme to havean average molecular weight of about 3,500) may be treated with thetarget under appropriate conditions to examine whether the dextrin ishydrolyzed. Those skilled in the art would design appropriate conditionsfor reaction with a target and procedures for determining whetherdextrin is hydrolyzed.

(Flavonoid Compounds)

As used herein, the term “flavonoid compound” is intended to includeboth flavonoid and esculetin, unless otherwise specified.

As used herein, the term “flavonoid” is intended to mean a catechincompound (flavanol), flavanone, flavone, flavonol, flavanonol,isoflavone, anthocyan or chalcone, as well as a methylated derivativethereof, unless otherwise specified. Flavonoid includes naringenin,quercetin, daidzein, genistein and kaempferol. Flavonoid available foruse in the present invention may be of natural or synthetic origin.

As used herein, the term “catechin compound” is used in a broad sense tomean a polyoxy derivative of 3-oxyflavan, unless otherwise specified.This includes catechin, gallocatechin and 3-galloyl derivatives thereof,as well as optical isomers ((+)-, (−)-, (+)-epi- and (−)-epi-isomers)and racemates thereof. Specific examples include catechin, gallocatechin(GC), catechin gallate (catechin-3-O-gallate; CG), gallocatechin gallate(gallocatechin-3-O-gallate; GCG), epicatechin (EC),, epigallocatechin(EGC), epicatechin gallate (epicatechin-3-O-gallate; ECG) andepigallocatechin gallate (epigallocatechin-3-O-gallate; EGCG), as wellas optical isomers thereof. Methylated derivatives of catechin compoundsrefer to derivatives of the above catechin compounds, in which H in atleast one OH group is replaced by methyl. Examples of methylatedderivatives of catechin compounds include those having methyl in placeof H in the OH group located at any of the 3′-, 4′-, 3″- and4″-positions of epicatechin, epigallocatechin, epicatechin gallate orepigallocatechin gallate. Catechin compounds and their methylatedderivatives available for use in the present invention may be of naturalor synthetic origin. Examples of natural origin include tea extracts,concentrated and purified products thereof (e.g., green tea extractssuch as Teavigo (DSM Nutrition Japan), Polyphenon (Mitsui Norin Co.,Ltd., Japan) and Sunphenon (Taiyo Kagaku Co., Ltd., Japan)), as well asextracts of a tea cultivar “Benifuki.”

In the present invention, flavonoid compounds may be used either aloneor in combination.

(Glycosyl Donors)

As used herein, the term “glycosyl donor” is intended to mean acarbohydrate which can serve as a substrate for an enzyme in the methodof the present invention and can be hydrolyzed to supply a sugar residueto a flavonoid compound, unless otherwise specified. Glycosyl donorsavailable for use in the present invention are carbohydrates containinga maltotriose residue, including maltotriose, maltotetraose,maltopentaose, maltohexaose, maltoheptaose, dextrin, γ-cyclodextrin andsoluble starch. As used herein, the term “dextrin” is intended to mean ahydrolysate of starch, unless otherwise specified, while the term“soluble starch” is intended to mean a hydrolysate of starch, which issoluble in hot water, unless otherwise specified. Hydrolysis may beaccomplished by using any means such as an acid, heat or an enzyme. Inthe present invention, it is possible to use a hydrolysate having anaverage molecular weight of about 3,500 as an example of dextrin and ahydrolysate having an average molecular weight of about 1,000,000 as anexample of soluble starch.

As used herein, the term “stringent conditions” refers to conditions of6 M urea, 0.4% SDS and 0.5×SSC, or hybridization conditions equivalentthereto, unless otherwise specified. If necessary, more stringentconditions (e.g., 6 M urea, 0.4% SDS and 0.1×SSC) or hybridizationconditions equivalent thereto may be applied in the present invention.Under each of these conditions, the temperature may be set to about 40°C. or higher. When more stringent conditions are required, thetemperature may be set to a higher value, for example about 50° C. andmore particularly about 65° C.

Moreover, the expression “nucleotide sequence comprising substitution,deletion, insertion and/or addition of one or several nucleotides” asused herein does not provide any limitation on the number of nucleotidesto be substituted, deleted, inserted and/or added, as long as a proteinencoded by a polynucleotide consisting of such a nucleotide sequence hasdesired functions. The number of such nucleotides is around 1 to 9 oraround 1 to 4, or alternatively, a larger number of nucleotides may besubstituted, deleted, inserted and/or added as long as such a mutationallows encoding of the same or a functionally similar amino acidsequence. Likewise, the expression “nucleotide sequence comprisingsubstitution, deletion, insertion and/or addition of one or severalamino acids as used herein does not provide any limitation on the numberof amino acids to be substituted, deleted, inserted and/or added, aslong as a protein having such an amino acid sequence has desiredfunctions. The number of such amino acids is around 1 to 9 or around 1to 4, or alternatively, a larger number of amino acids may besubstituted, deleted, inserted and/or added as long as such a mutationprovides a functionally similar amino acid. Means for preparing apolynucleotide or protein having such a nucleotide or amino acidsequence are well known to those skilled in the art.

As used herein to describe an amino acid sequence, the term “highidentity” refers to a sequence identity of at least 60% or more,preferably 70% or more, more preferably 80% or more, even morepreferably 90% or more, and most preferably 95% or more.

Search and analysis for identity between nucleotide or amino acidsequences may be accomplished by using any algorithm or program (e.g.,BLASTN, BLASTP, BLASTX, ClustaiW) well known to those skilled in theart. In the case of using a program, parameters may be set as requiredby those skilled in the art, or alternatively, default parametersspecific for each program may be used. Detailed procedures for suchanalysis are also well known to those skilled in the art.

The polynucleotide of the present invention can be obtained from naturalproducts by using techniques such as hybridization and polymerase chainreaction (PCR).

The polynucleotide of the present invention is preferably derived fromthe genus Trichoderma, more preferably Trichoderma viride.

The present invention also provides a recombinant vector carrying thepolynucleotide of the present invention, as well as a transformant(e.g., a transformed E. coli, yeast or insect cell) transformed with therecombinant vector. The present invention further provides atransformation method comprising the step of transforming a host (e.g.,an E. coli, yeast or insect cell) by using the polynucleotide of thepresent invention (e.g., the step of transforming a host with therecombinant vector of the present invention).

There is no particular limitation on the vector into which thepolynucleotide of the present invention is inserted, as long as itallows expression of the insert in a host. Such a vector generally has apromoter sequence, a terminator sequence, a sequence for inducibleexpression of an insert in response to external stimulation, a sequencerecognized by a restriction enzyme for insertion of a target gene, and asequence encoding a marker for transformant selection. To create such arecombinant vector and to effect transformation with such a recombinantvector, techniques well known to those skilled in the art may beapplied.

[Glycosyltransferase Proteins]

The present invention also provides a novel glycosyltransferase proteinand a homolog thereof, i.e., a protein comprising (i), (j) or (k) shownbelow (preferably a protein consisting of (l), (j) or (k) shown below):

(i) a protein which consists of the amino acid sequence shown in SEQ IDNO: 10;

(j) a protein which consists of an amino acid sequence comprisingsubstitution, deletion, insertion and/or addition of one or severalamino acids in the amino acid sequence shown in SEQ ID NO: 10 and whichhas glycosylation activity on a flavonoid compound; or

(k) a protein which consists of an amino acid sequence sharing anidentity of at least 60% or more with the amino acid sequence shown inSEQ ID NO: 10 and which has glycosylation activity on a flavonoidcompound.

The present invention also provides a mature protein of the above novelglycosyltransferase protein and a homolog thereof, which is modified toremove a putative secretion signal sequence region, i.e., a proteincomprising (p), (q) or (r) shown below (preferably a protein consistingof (p), (q) or (r) shown below):

(p) a protein which consists of the amino acid sequence shown in SEQ IDNO: 26;

(q) a protein which consists of an amino acid sequence comprisingsubstitution, deletion, insertion and/or addition of one or severalamino acids in the amino acid sequence shown in SEQ ID NO: 26 and whichhas glycosylation activity on a flavonoid compound; or

(r) a protein which consists of an amino acid sequence sharing anidentity of at least 60% or more with the amino acid sequence shown inSEQ ID NO: 26 and which has glycosylation activity on a flavonoidcompound.

The present invention also provides a protein having glycosylationactivity on a flavonoid compound, which is encoded by a polynucleotidebeing derived from the genus Trichoderma (preferably Trichoderma viride,Trichoderma reesei, Trichoderma saturnisporum, Trichoderma ghanense,Trichoderma koningii, Trichoderma hamatum, Trichoderma harzianum orTrichoderma polysporum) and comprising any one of the nucleotidesequences shown in SEQ ID NOs: 11 to 24. A preferred example of such aprotein is a protein having glycosylation activity on a flavonoidcompound, which is encoded by a polynucleotide comprising any one of thenucleotide sequences shown in SEQ ID NOs: 11 to 24 and sharing highidentity with the nucleotide sequence shown in SEQ ID NO: 6, 9 or 25.

The protein of the present invention can be isolated and purified from aculture supernatant obtained by culturing a species belonging to thegenus Trichoderma, such as Trichoderma viride, Trichoderma reesei,Trichoderma saturnisporum, Trichoderma ghanense, Trichoderma koningii,Trichoderma hamatum, Trichoderma harzianum or Trichoderma polysporum ina known culture solution. Alternatively, the protein of the presentinvention can also be obtained as a recombinant protein from atransformant (e.g., a transformed yeast or E. coli cell) transformedwith a recombinant vector carrying the polynucleotide of the presentinvention.

[Enzymological Properties of Enzyme TRa2]

Among novel enzyme proteins provided by the present invention,particularly preferred is a glycosyltransferase consisting of the aminoacid sequence shown in SEQ ID NO: 10 (herein also referred to as“TRa2”), which is obtained from the culture supernatant of Trichodermaviride strain IAM5141. This enzyme has the following enzymologicalfeatures in reaction between flavonoid and glycosyl donor.

Glycosyl Donor Selectivity:

Under the conditions shown in the Example section, this enzyme usesmaltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose,soluble starch, dextrin, γ-cyclodextrin or the like as a glycosyl donor,but does not target cellobiose, dextran, maltose monohydrate,carboxymethylcellulose sodium salt, isomaltooligosaccharide,α-cyclodextrin, β-cyclodextrin or the like as a glycosyl donor.Moreover, this enzyme is a glycosyltransferase capable of producing notonly sugars composed of one or two glucose molecules, but alsoglycosides whose sugar chain length is three (G3) or more glucosemolecules.

Substrate Specificity:

This enzyme can act on and glycosylate a wide range of polyphenolsincluding major flavonoid members such as catechin, epigallocatechingallate, naringenin, quercetin, daidzein, genistein and kaempferol, aswell as esculetin.

Optimum Reaction Temperature and pH (Reaction Temperature Dependence andpH Dependence):

In the case of hydrolysis reaction, this enzyme allows a satisfactoryreaction at a temperature of about 20° C. to about 60° C., particularlyabout 35° C. to about 55° C., or at a pH of about 4.0 to about 7.0,particularly about 4.5 to about 5.5, under the conditions shown in theExample section.

Likewise, in the case of glycosyltransferase reaction, this enzymeallows a satisfactory reaction at a temperature of about 20° C. to about60° C., particularly about 45° C. to about 55° C., or at a pH of about4.0 to about 9.0, particularly about 4.5 to about 7.0, under theconditions shown in the Example section.

Temperature Stability and pH Stability:

Under the conditions shown in the Example section, this enzyme is notdeactivated either by treatment at about 20° C. to about 45° C. for 1 hror by treatment at about pH 5.0 to 9.0 at room temperature for 16 hr.

[Method for Preparing a Glycoside of a Flavonoid Compound]

The present invention also provides a method for preparing a glycosideof a flavonoid compound, which comprises allowing the novelglycosyltransferase of the present invention to act on a mixture of theflavonoid compound and a glycosyl donor (preferably dextrin, solublestarch or γ-cyclodextrin).

Glycosides of flavonoid compounds provided by the present invention havethe following formula:

wherein

at least one of R^(I) to R⁵ represents a sugar residue, and each of theothers represents OH or OCH₃, or

at least one of le to R⁴ represents a sugar residue and each of theothers represents OH or OCH₃, and R⁵ represents H; and

X represents H, CH₃, a galloyl group or a methylated galloyl group.

This formula further encompasses the glycosides of flavonoid compoundslisted below:

5-O-(4-O-α-D-glucopyranosyl-α-D-glucopyranosyl)-(+)-catechln;

5-O-α-D-glucopyranosyl-(+)-catechin;

4′-O-(4-O-α-D-glucopyranosyl-α-D-glucopyranosyl)-(+)-catechin;

4′-O-α-D-glucopyranosyl-(+)-catechin;

3′-O-(4-O-α-D-glucopyranosyl-α-D-glucopyranosyl)-(+)-catechin;

3′-O-α-D-glucopyranosyl-(+)-catechin;

7-O-α-D-glucopyranosyl-(−)-epigallocatechin-3-O-gallate;

7-O-(4-O-α-D-glucopyranosyl-α-D-glucopyranosyl)-(−)-epigallocatechin-3-O-gallate;

3′-O-α-D-glucopyranosyl-(−)-epigallocatechin-3-O-gallate; and

3′-O-(4-O-α-D-glucopyranosyl-α-D-glucopyranosyl)-(−)-epigallocatechin-3-O-gallate,as well as optical isomers thereof.

[Uses of Glycosides]

Glycosides obtained by the present invention can be used as foodcompositions, pharmaceutical compositions or cosmetic compositions. Morespecifically, for example, such a composition incorporating a glycosideof a catechin compound can be used as an agent for the followingpurposes, as in the case of catechin: anti-allergy, anti-oxidation,anti-cancer, anti-inflammation, anti-bacteria/anti-caries, anti-virus,detoxication, intestinal flora improvement, odor elimination,anti-hypercholesterolemia, anti-hypertension, anti-hyperglycemia,anti-thrombosis, dementia prevention, body fat burning, inhibition ofbody fat accumulation, endurance improvement, anti-fatigue or renalfunction improvement, or alternatively, can also be used as a foodcomposition, a pharmaceutical composition or a cosmetic composition.

Food compositions include nutritional supplementary foods, health foods,therapeutic dietary foods, general health foods, supplements andbeverages. Beverages include tea beverages, juices, soft drinks, anddrinkable preparations. Pharmaceutical compositions may be prepared asdrugs or quasi drugs, preferably oral formulations or dermatologicexternal preparations, and may be provided in the form of solutions,tablets, granules, pills, syrups, lotions, sprays, plasters orointments. Cosmetic compositions may be provided in the form of creams,liquid lotions, emulsion lotions or sprays.

The amount of glycoside(s) incorporated into the food, pharmaceutical orcosmetic composition of the present invention is not limited in any wayand may be determined as required by those skilled in the art inconsideration of, e.g., solubility and taste by referring to preferreddaily intakes of the corresponding flavonoid compound(s). For example,the amount of the glycoside(s) of the present invention incorporatedinto a composition may be set to 0.01% to 99.9% by weight or may bedetermined such that the glycoside(s) of the present invention can begiven 100 mg to 20 g per day as a single dose or in divided doses (e.g.,three doses).

The food, pharmaceutical or cosmetic composition of the presentinvention may further comprise various ingredients acceptable for food,pharmaceutical or cosmetic purposes. Examples of these additives and/oringredients include vitamins, saccharides, excipients, disintegratingagents, binders, lubricants, emulsifiers, isotonizing agents, buffers,solubilizers, antiseptics, stabilizers, antioxidants, coloring agents,correctives, flavorings, coagulating agents, pH adjustors, thickeners,tea extracts, herbal extracts, and minerals.

Other Embodiments

In the present invention, a glycosyltransferase protein can beimmobilized on an appropriate carrier for use as an immobilized enzyme.As a carrier, any conventional resin used for the same purpose may beused, including basic resins (e.g., MARATHON WBA (Dow Chemical), SAseries, WA series or FP series (Mitsubishi Chemical Corporation, Japan),and Amberlite IRA904 (Organo)), as well as hydrophobic resins (e.g.,Diaion FPHA13 (Mitsubishi Chemical Corporation, Japan), HP series(Mitsubishi Chemical Corporation, Japan), and Amberlite XAD7 (Organo)).In addition, other resins such as Express-Ion D (Whatman),DEAE-Toyopearl 650M (Tosoh Corporation, Japan) and DEAE-sepharose CL4B(Amersham Biosciences) may be preferred for use. Any conventionaltechnique can be used for enzyme immobilization, as exemplified byphysical adsorption, the binding method which uses ionic or covalentbinding for immobilization, the crosslinking method which uses a reagenthaving a divalent functional group for immobilization throughcrosslinking, and the entrapping method which embeds an enzyme within agel or semipermeable membrane of network structure. For example,immobilization may be accomplished by allowing an enzyme (20 to 2,000mg, e.g., 50 to 400 mg) in distilled water to be adsorbed to 5 ml ofeach resin, followed by removal of the supernatant and drying.

The present invention also provides a method for determining thenucleotide sequence of a polynucleotide encoding a protein havingglycosylation activity on a flavonoid compound or the amino acidsequence of a protein having glycosylation activity on a flavonoidcompound, which comprises using all or part of the nucleotide or aminoacid sequence shown in any one of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO:10 and SEQ ID NOs: 11 to 26.

Advantages of the Invention

The present invention provides novel enzymes which allow efficientglycosylation of flavonoid, as well as genes thereof.

The novel enzymes of the present invention allow glycosylation offlavonoid compounds to thereby improve their water solubility. Thissuggests that the present invention can enhance the oral absorption offlavonoid compounds. Moreover, improved water solubility will contributeto not only improvement of dissolution rate in water, but alsoimprovement of absorption rate in the body. Thus, the present inventionallows flavonoid compounds to exert their useful activity (e.g.,antioxidative activity) in vivo with high efficiency.

The present invention can also modify the taste of flavonoid compoundsthrough glycosylation. Particularly when a flavonoid compound havingbitter and astringent tastes like a catechin compound is glycosylated inaccordance with the present invention, such tastes can be reduced.

The present invention can also improve the heat stability of flavonoidthrough glycosylation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an HPLC analysis chart of catechin when treated with acrude enzyme solution of Trichoderma viride IAM5141.

FIG. 2 shows a dendrogram prepared by a dendrogram preparation program,Tree view, with respect to the amino acid sequences of putative ORFshaving the alpha-amylase catalytic domain (accession No. PF00128) motifextracted from the genomic information databases of Aspergillusnidulans, Neurospora crassa, Magnaporthe grisea and Fusariumgraminearum.

FIG. 3 shows an alignment of 4 amino acid sequences in Group 1 of FIG.2, along with their highly conserved regions (underlined).

FIG. 4 shows a comparison between genomic DNA sequence (SEQ ID NO: 6)and cDNA sequence (SEQ ID NO: 9) of TRa2.

FIG. 5 shows the cDNA nucleotide sequence of TRa2 and its correspondingdeduced amino acid sequence. The double-underlined part represents aputative secretion signal sequence.

FIG. 6 shows a comparison of the primary structure between the deducedamino acid sequence of TRa2 and the Taka-amylase precursor amino acidsequence (GB No. BAA00336). Underlined: putative secretion signal ofTRa2; broken-underlined: secretion signal of Taka-amylase;double-underlined: 4 regions highly conserved among α-amylase familyenzymes; and amino acid residues indicated with *: amino acid residueslocated at catalytic sites.

FIG. 7 shows HPLC analysis charts of the reaction solution when(+)-catechin or (−)-epigallocatechin-3-O-gallate and dextrin were addedto and reacted in a culture supernatant stock of T. viride IAM5141 or aconcentrate thereof.

FIG. 8 shows the results of partial nucleotide sequence alignment forTRa2 homologs obtained from genomic DNAs prepared from various strainsof the genus Trichoderma.

FIG. 9 is a graph showing glycosylation activity of a crude TRa2 enzymesolution prepared from the culture supernatant of a transformant (strainTRa2-1), when used for reaction between each glycosyl acceptor compound((+)-catechin, (−)-epigallocatechin-3-O-gallate, esculetin, naringenin,quercetin, daidzein, genistein or kaempferol) and dextrin.

FIG. 10 is graphs showing the temperature stability and pH stability ofTRa2.

FIG. 11 is graphs showing the temperature dependence and pH dependenceof TRa2-catalyzed carbohydrate hydrolysis reaction.

FIG. 12 is graphs showing the temperature dependence and pH dependenceof TRa2-catalyzed glycosylation reaction.

FIG. 13 is a graph showing the % remaining of (+)-catechin or4′-O-α-D-glucopyranosyl-(+)-catechin after a solution containing thesame was treated at different temperatures ranging from 4° C. to 100° C.for 0 to 4 hours.

FIG. 14 is a graph showing the solubility of (+)-catechin and5-O-α-D-glucopyranosyl-(+)-catechin in water.

EXAMPLES Example 1 Catechin Glycosylation Activity in TrichodermaCulture

Trichoderma viride strain IAM5141 was inoculated from a slant into aliquid medium (10 ml) containing 1% yeast extract (Difco), 1%polypeptone (Nihon Pharmaceutical Co., Ltd., Japan) and 2% dextrin(Nacalai Tesque, Inc., Japan), followed by shaking culture at 30° C. for1 day to give a pre-cultured solution. Further, the entire volume of thepre-cultured solution was inoculated into 900 ml of the same liquidmedium and cultured at 30° C. for 3 days, followed by filter filtrationto prepare a culture supernatant solution. After addition of ammoniumsulfate (387 g, 80% saturation) to the culture supernatant (690 ml), themixture was stirred and centrifuged to collect a precipitate. Theresulting precipitate was diluted with 10 ml of 0.1 M acetate buffer (pH5.0) for use as a crude enzyme solution.

To the crude enzyme solution (100 μl), catechin (3 mg) and dextrin (10mg) were added and stirred at 50° C. for 24 hours to cause an enzymereaction. The reaction solution was diluted 10-fold with 0.1%trifluoroacetic acid (TFA), 10 μl of which was then analyzed by highperformance liquid chromatography (HPLC).

Analysis Conditions

Column: Develosil C30-UG-5 (4.6×150 mm)

Gradient conditions: 5% Eluent B→50% Eluent B/20 min

Eluent A: 0.1% TFA/distilled water

Eluent B: 90% acetonitrile/0.08% TFA

Flow rate: 1 ml/min

Detection wavelength: 280 nm

As shown in FIG. 1, the results confirmed the generation of a catechinglycoside through the above reaction. Moreover, it was also confirmedthat a similar glycoside was generated in the case of usingγ-cyclodextrin as a glycosyl donor. These results suggest that T. viridestrain IAM5141 produces and secretes an enzyme which glycosylatescatechin using dextrin or γ-cyclodextrin as a glycosyl donor.

Example 2 PCR Cloning of Partial α-amylase Homolog Sequences

In view of the facts that dextrin and y-cyclodextrin are polymers inwhich glucose residues are linked through α-1,4 linkages and that theintended enzyme has the ability to degrade these polymers, it issuggested that the enzyme may be an α-amylase family-like enzyme.

For further study, with respect to a putative ORF having thealpha-amylase catalytic domain (accession No. PF00128) motif in theprotein family database (PFAM), 9, 6, 8 and 6 amino acid sequences wereextracted from the genomic information databases of Aspergillusnidulans, Neurospora crassa, Magnaporthe grisea and Fusariumgraminearum, respectively, among microorganisms belonging to the sameascomycetous filamentous fungi as Trichoderma and already identified fortheir genome sequences. For these sequences, an alignment was preparedby the homology search program ClustalW and a dendrogram was prepared bythe dendrogram preparation program Tree view, whereby the sequences weregrouped on the basis of their homology. Four amino acid sequences inGroup 1 of FIG. 2, i.e., MG02772.4 (EAA47529), MG10209.4 (EAA48146),AN3388.2 (EAA63356) and FG03842.1 (EAA71544) (numbers in parentheses areGenebank Accession Nos.) were aligned to synthesize oligo DNAscorresponding to the amino acid sequences of their highly conservedregions (FIG. 3, underlined).

AMY-12f: (SEQ ID NO: 1) 5′-TAYTGYGGNGGNACNTTYAARGGNYT-3′ AMY-15r:(SEQ ID NO: 2) 5′-TTYTCNACRTGYTTNACNGTRTCDAT-3′ AMY-17r: (SEQ ID NO: 3)5′-GGTNAYRTCYTCNCKRTTNGCNGGRTC-3′

From wet cells (about 1 g) of T. viride IAM5141 curtured as describedabove, genomic DNA was extracted with a DNeasy plant Maxi Kit (QIAGEN).This genomic DNA (50 ng) was used as a template to perform PCR reactionwith primers AMY-12f and AMY-15r or primers AMY-12f and AMY-17r. Namely,PCR was accomplished by using ExTaq (Takara Bio Inc., Japan) under thefollowing conditions: 94° C. for 2 minutes, (94° C. for 1 minute, 50° C.for 1 minute, 72° C. for 1 minute)×30 cycles, and 72° C. for 10 minutes.The PCR products were analyzed by agarose gel electrophoresis,confirming a fragment of approximately 0.6 kbp for the primercombination of AMY-12f and AMY-15r and a fragment of approximately 1.0kbp for the primer combination of AMY-12f and AMY-17r. Then, these DNAfragments were excised from the agarose gel and purified with a GFX PCRDNA and Gel Band Purification Kit (Amersham Biosciences). Each DNA wascloned with a TOPO-TA cloning kit (Invitrogen) and analyzed for itsnucleotide sequence using an ABI 3100 Avant (Applied Biosystems). Thenucleotide sequence obtained for the former fragment was included withinthe nucleotide sequence obtained for the latter fragment. A homologysearch with Blastx was made for this nucleotide sequence against aminoacid sequences registered in GenBank, indicating that the highesthomology was observed with MG10209.4 (EAA48146).

Example 3 Genome Sequence Determination of α-amylase Homolog

On the basis of the resulting nucleotide sequence of approximately 1.0kbp, the following primers were designed and used to perform InversePCR.

TRa2-2: (SEQ ID NO: 4) 5′-CCAACCTGGTATCTACATAC-3′ TRa2-3: (SEQ ID NO: 5)5′-AGATGGCATCAAATCCCAT-3′

First, the genomic DNA prepared from T. viride IAM5141 was completelydigested with Hindlil or PstI, and then closed by self-ligation throughovernight incubation at 16° C. with ligation high (Toyobo Co., Ltd.,Japan). These DNAs (0.1 μg each) were each used as a template to performPCR reaction with the above primers TRa2-2 and TRa2-3. PCR wasaccomplished by using LA Taq (Takara Bio Inc., Japan) under thefollowing conditions: 94° C. for 2 minutes, (95° C. for 30 seconds, 66°C. for 15 minutes)×30 cycles, and 72° C. for 10 minutes. The resultingPCR products were analyzed by agarose gel electrophoresis, confirming aDNA fragment of approximately 2 kb for the case of using theHindlil-digested genomic DNA as a template, and a DNA fragment ofapproximately 4.5 kb for the case of using the PstI-digested genome as atemplate. These DNA fragments were each excised from the agarose gel andcloned in the same manner as described above. Nucleotide sequences weredetermined from both ends of the inserted fragments. The nucleotidesequences from the HindIII-digested genome and the PstI-digested genomewere found to overlap with each other until reaching the restrictionenzyme sites. The nucleotide sequences thus obtained were ligated to thepartial sequence previously obtained. This nucleotide sequence is shownin FIG. 4 (TRa2-gDNA) and SEQ ID NO: 6. The coding region of theα-amylase homolog was deduced by comparison with the 4 sequences inGroup 1 of FIG. 2, appearance of an initiation codon and a terminationcodon, etc. The initiation codon was considered to be ATG at nucleotides423-425, while the termination codon was considered to be TAA atnucleotides 1926-1928.

Example 4 cDNA Cloning of α-amylase homolog

From the cells of T. viride strain IAM5141 curtured as describedabove(about 0.1 g), total RNA was extracted with an RNeasy plant minikit. The total RNA (1 μg) was used for cDNA synthesis in a SuperScriptFirst-Strand system for RT-PCR(Invitrogen) using random hexamers.

On the basis of the genome sequence previously obtained, the followingprimers were designed.

TRa2EcoRI-f2: (SEQ ID NO: 7) 5′-GGAATTCATGAAGCTTCGATCCGCCGTCCC-3′TRa2XhoI-r2: (SEQ ID NO: 8) 5′-CCGCTCGAGTTATGAAGACAGCAGCACAAT-3′

The synthesized cDNA was used as a template to perform PCR reaction withthe above primers TRa2EcoRI-f2 and TRa2XhoI-r2. PCR was accomplished byusing Ex Taq (Takara Bio Inc., Japan) under the following conditions:94° C. for 2 minutes, (94° C. for 1 minute, 55° C. for 1 minute, 72° C.for 2 minutes) x 30 cycles, and 72° C. for 10 minutes. The resulting PCRproducts were analyzed by agarose gel electrophoresis, confirming a DNAfragment of approximately 1.5 kb. This DNA fragment was excised from theagarose gel and purified by GFX. The resulting DNA fragment was clonedwith a TOPO-TA cloning kit (Invitrogen) to construct plasmidpCRTRa2-cDNA, and the nucleotide sequence of the cDNA was determined(FIG. 4, FIG. 5 and SEQ ID NO: 9). The genomic DNA sequence previouslyobtained was compared with the cDNA sequence thus obtained, indicatingthat the genome sequence contained two introns (FIG. 4). The cDNAsequence was found to contain 1392 by ORF encoding a protein composed of463 amino acid residues (FIG. 5 and SEQ ID NO: 10). This gene wasdesignated as TRa2. When the deduced amino acid sequence encoded by thisgene was analyzed by Signal P (Nielsen H. et.al., Protein Eng., 10, 1-6,1997), the N-terminal 20 amino acid residues appeared to constitute asecretion signal sequence. Further, a homology search was made for thededuced amino acid sequence encoded by TRa2 in the same manner asdescribed above, indicating that the highest homology was observed withAN3388.2 (EAA63356). The deduced amino acid sequence of TRa2 protein wascompared with the amino acid sequence of Taka-amylase, which is a knownα-amylase. The result indicated that 4 conserved regions among a-amylasefamily enzymes were also conserved in this enzyme (FIG. 6,double-underlined), and that the aspartic acid residue, the glutamicacid residue and the aspartic acid residue, each serving as an activecenter, were all conserved (FIG. 6, amino acid residues indicated with*).

Example 5 Construction of Secretory Expression System for TRa2 Proteinin Yeast

The plasmid pCRTRa2-cDNA was digested with restriction enzymes EcoRI andXhoI to give a fragment of approximately 1.5 kb, which was then ligatedto an EcoRI- and SalI-digested fragment of plasmid pYE22m (Biosci.Biotech. Biochem., 59(7), 1221-1228, 1995) using ligation high (ToyoboCo., Ltd., Japan) to thereby obtain plasmid pYETRa2.

The plasmid pYETRa2 was used to transform yeast S. cerevisiae strainEH1315 by the lithium acetate method. The resulting transformed strainwas designated as strain TRa2-1. A loopful of the strain TRa2-1 wasinoculated into 10 ml YPD (Difco) liquid medium and cultured withshaking at 30° C. for 2 days. Since the TRa2 protein has a secretionsignal sequence composed of 20 amino acid residues at its N-terminalend, the protein was considered to be secreted into a culture solution.Then, the yeast cells were precipitated by centrifugation to collect theculture supernatant.

Example 6 Measurement of Glycosidase Activity of TRa2

The culture supernatant (500 μl) was concentrated about 5-fold usingMicrocon YM-30 (Amicon). The above concentrate (10 μl) was added to 1001of 20 mM acetate buffer (pH 5.0) containing 0.5% maltose, maltotriose,maltotetraose, dextrin, α-cyclodextrin, β-cyclodextrin orγ-cyclodextrin, and reacted at 50° C. for 1 hour.

After completion of the reaction, each sample was analyzed by TLC asfollows. The plate used was a silica gel G-60 plate (Merck & Co., Inc.),and the developing solution used was 2-propanol:acetone:0.5 M lacticacid=2:2:1. For detection, the plate was sprayed with sulfuricacid:ethanol=1:9, air-dried and then heated on a hot plate. As a result,none of the sugars was degraded in a culture solution of the controlstrain (strain C-1) transformed with vector pYE22m. In contrast, in aculture solution of the strain TRa2-1, maltotriose, maltotetraose,dextrin and γ-cyclodextrin were degraded to mainly generate maltose andglucose, but there was no degradation of maltose, α-cyclodextrin andβ-cyclodextrin.

Example 7 Measurement of Glycosylation Activity of TRa2

To 100 μl of a culture supernatant stock or a concentrate thereofconcentrated about 5-fold with a VIVASPIN 10,000 MWCO/PES (VIVASCIENCE),(+)-catechin or (−)-epigallocatechin-3-O-gallate (3 mg) and dextrin (10mg) were added and reacted with stirring at 50° C. for 1 day. Aftercompletion of the reaction, the reaction solution was diluted 10-foldwith a 0.1% trifluoroacetic acid solution and analyzed by highperformance liquid chromatography (HPLC) under the same conditions asused in Example 1. As a result, no reaction product was observed in thereaction solution reacted with the culture supernatant from the controlstrain (strain C-1) transformed with vector pYE22m, whereas thegeneration of catechin glycosides and epigallocatechin-3-O-gallategycosides was confirmed in the case of the strain TRa2-1 (FIG. 7).

Example 8 TRa2-Catalyzed Preparation of Catechin Glycosides

The strain TRa2-1 was inoculated into 200 ml YPD liquid medium andcultured with shaking at 30° C. for 3 days. The cells were collected bycentrifugation to obtain the culture supernatant. This culturesupernatant (100 ml) was concentrated to 50 ml using a ultrafiltrationdisk NMWL 30000/regenerated cellulose while adding 100 ml of 0.1 Macetate buffer (pH 5), and used as a TRa2 enzyme solution.

The above TRa2 enzyme solution (50 ml) was mixed with (+)-catechin (1.5g) and dextrin (5 g), followed by stirring at 45° C. for 18 hr. Thereaction solution was centrifuged, and the supernatant was adsorbed ontoa LH2O (Amersham Biosciences) resin 60 ml φ2.5×20 cm column. Afterelution with distilled water (120 ml) and 10% ethanol (240 ml),glycoside fractions were collected and lyophilized to give 530 mglyophilized powder, 50 mg of which was then dissolved in 5 ml distilledwater and separated on a Develosil C30-UG-5 column 20×250 mm, A: 0.1%TFA/distilled water, B: 90% methanol/0.1% TFA, 30% B, 3 ml/min, 280 nm.Peaks 1 to 6 were collected and lyophilized in the order in which theywere eluted from the HPLC column. MS and NMR analyses suggested thatPeak 1 was 5-O-(4-O-α-D-glucopyranosyl-α-D-glucopyranosyl)-(+)-catechin,Peak 2 was 5-O-α-D-glucopyranosyl-(+)-catechin, Peak 3 was4′-O-(4-O-α-D-glucopyranosyl-α-D-glucopyranosyl)-(+)-catechin, Peak 4was 4′-O-α-D-glucopyranosyl-(+)-catechin, Peak 5 was3′-O-(4-O-α-D-glucopyranosyl-α-D-glucopyranosyl)-(+)-catechin, and Peak6 was 3′-O-α-D-glucopyranosyl-(+)-catechin.

5-O-(4-O-α-D-glucopyranosyl-α-D-glucopyranosyl)-(+)-catechin: m/z 615.2,NMR δ ppm (D₂O); 2.71 (1H, dd), 2.85 (1H, dd), 3.42 (1H, t), 3.56-3.85(9H, m), 4.19 (1H, t), 4.26 (1H, dd), 4.87 (1H, d), 5.70 (1H, d), 6.19(1H, d), 6.39 (1H, d), 6.83 (1H, dd), 6.90-6.93 (2H, m).

5-O-α-D-glucopyranosyl-(+)-catechin: m/z 453.2, NMR δ ppm (D₂O); 2.62(1H, dd), 2.81 (1H, dd), 3.43 (1H, t), 3.45-3.55 (1H, m), 3.6-3.7 (3H,m), 3.83 (1H, t), 4.18 (1H, dd), 4.76 (1H, d), 5.61 (1H, d), 6.09 (1H,d), 6.31 (1H, d), 6.77 (1H, d), 6.8-6.9 (2H, m).

4′-O-(4-O-α-D-glucopyranosyl-α-D-glucopyranosyl)-(+)-catechin: m/z615.2, NMR 8 ppm (D₂O); 2.54 (1H, dd), 2.81 (1H, dd), 3.43 (1H, t), 3.60(1H, dd), 3.68-3.94 (9H, m), 4.19-4.28 (2H, m), 4.82 (1H, d), 5.44 (1H,d), 5.62 (1H, d), 6.04 (1H, d), 6.11 (1H, d), 6.91 (1H, dd), 7.00 (1H,d), 7.22 (1H, d).

4′-O-α-D-glucopyranosyl-(+)-catechin: m/z 453.2, NMR δ ppm (D₂O): 2.45(1H, dd), 2.73 (1H, dd), 3.45 (1H, t), 3.65-3.75 (4H, m), 4.11 (1H, dd),4.7-4.75 (2H, m), 5.53 (1H, d), 5.95 (1H, d), 6.02 (1H, d), 6.83 (1H,dd), 6.91 (1H, d), 7.15 (1H, d).

3′-O-(4-O-α-D-glucopyranosyl-α-D-glucopyranosyl)-(+)-catechin: m/z615.2, NMR 8 ppm (D₂O); 2.54 (1H, dd), 2.80 (1H, dd), 3.44 (1H, t), 3.59(1H, dd), 3.67-3.90 (9H, m), 4.17-4.24 (2H, m), 4.83 (1H, d), 5.41 (1H,d), 5.55 (1H, d), 6.03 (1H, d), 6.10 (1H, d), 7.10 (1H, d), 7.06 (1H,d), 7.26 (1H, d).

3′-O-α-D-glucopyranosyl-(+)-catechin: m/z 453.2, NMR δ ppm (D₂O); 2.43(1H, dd), 2.73 (1H, dd), 3.27 (1H, s), 3.44 (1H, t), 3.6-3.7 (4H, m),3.88 (1H, t), 4.10 (1H, dd), 4.69 (1H, d), 5.46 (1H, d), 5.93 (1H, s),6.01 (1H, s), 6.89 (1H, d), 6.94 (1H, dd), 7.18 (1H, d).

Example 9 TRa2-Catalyzed Preparation of Epigallocatechin-3-O-gallateGlycosides

The strain TRa2-1 was inoculated into 100 ml YPD liquid medium andcultured with shaking at 30° C. for 3 days. The cells were collected bycentrifugation to obtain the culture supernatant. This culturesupernatant (45 ml) was concentrated to 20 ml using a ultrafiltrationdisk NMWL 30000/regenerated cellulose while adding 50 ml of 0.1 Macetate buffer (pH 5), and used as a TRa2 enzyme solution. This TRa2enzyme solution (20 ml) was mixed with (−)-epigallocatechin-3-O-gallate(600 mg) and dextrin (2 g), followed by stirring at 50° C. for 1 day.The reaction solution was centrifuged, and the supernatant was adsorbedonto a LH20 resin 25 ml/41.5×30 cm column. After elution with distilledwater (100 ml), 10% ethanol (100 ml), 20% ethanol (100 ml) and 30%ethanol (200 ml), the 30% ethanol fraction was collected andlyophilized. The lyophilized powder (120 mg) was dissolved in 12 mldistilled water and separated on a Develosil C30-UG-5 column 20 x 250mm, A: 0.1% TFA/distilled water, B: 90% methanol/0.1% TFA, 40% B, 3ml/min, 280 nm. MS and NMR analyses suggested that Peak 2 was7-O-(4-O-α-D-glucopyranosyl-α-D-glucopyranosyl)-(−)-epigallocatechin-3-O-gallate,Peak 5 was3′-O-(4-O-α-D-glucopyranosyl-α-D-glucopyranosyl)-(−)-epigallocatechin-3-O-gallate,and Peak 6 was 3′-O-α-D-glucopyranosyl-(−)-epigallocatechin-3-O-gallate.In contrast, Peak 3 was suggested to be a mixture of glucoside andmaltotetraoside, as judged by its MS data (m/z 621.2, 1107.3), and theglucoside was considered to be7-O-α-D-glucopyranosyl-(−)-epigallocatechin-3-O-gallate, as judged byits retention time.

7-O-(4-O-α-D-glucopyranosyl-α-D-glucopyranosyl)-(−)-epigallocatechin-3-O-gallate:m/z 783.2, NMR δ ppm (CD₃OD); 2.88 (1H, dd), 2.01 (1H, dd), 3.26 (1H,t), 3.46 (1H, dd), 3.6-3.9 (9H, m), 4.08 (1H, t), 5.00 (1H, s), 5.20(1H, d), 5.43 (1H, d), 5.54 (1H, s), 6.27 (1H, d), 6.34 (1H, d), 6.51(2H, d), 6.94 (2H, d).

3′-O-α-D-glucopyranosyl-(−)-epigallocatechin-3-O-gallate: m/z 621.1, δppm (CD3OD); 2.88 (1H, dd), 2.99 (1H, dd), 3.42 (1H, dd), 3.51 (1H, t),3.69 (1H, m), 3.8-3.9 (3H, m), 4.88 (1H, d), 4.98 (1H, s), 5.49 (1H,broad s), 5.95 (1H, d), 5.96 (1H, d), 6.65 (1H, d), 7.01 (2H, s), 7.11(1H, d).

3′-O-(4-O-α-D-glucopyranosyl-α-D-glucopyranosyl)-(−)-epigallocatechin-3-O-gallate:m/z 783.2, δ ppm (CD3OD); 2.87 (1H, broad d), 2.99 (1H, dd), 3.27 (1H,t), 3.44-3.48 (2H, m), 3.6-3.8 (4H, m), 3.85 (2H, d), 3.98 (1H, dd),4.06 (H, t), 4.85 (1H, d), 4.99 (1H, s), 5.28 (1H, d), 5.49 (1H, broads), 5.94 (1H, d), 5.96 (1H, d), 6.64 (1H, d), 7.01 (2H, s), 7.09 (1H,d).

Example 10 Construction of Yeast Surface Expression System for TRa2Protein

To express and display a TRa2 protein on the yeast surface, thefollowing vector was constructed. The plasmid pCRTRa2-cDNA was used as atemplate to perform PCR reaction with primers TraEcoRI-f2 andTRa2XhoI-r3. PCR was accomplished by using Ex Taq (Takara Bio Inc.,Japan) under the following conditions: 94° C. for 2 minutes, (94° C. for1 minute, 58° C. for 1 minute, 72° C. for 2 minutes)×25 cycles, and 72°C. for 10 minutes. The resulting PCR products were analyzed by agarosegel electrophoresis, confirming a DNA fragment of approximately 1.5 kb.

This DNA fragment was excised from the agarose gel and purified by GFX.The resulting DNA fragment was cloned with a TOPO-TA cloning kit(Invitrogen), confirmed for its nucleotide sequence, and designated asplasmid pCRTRa2-cDNA 2. The plasmid pCRTRa2-cDNA2 was digested withEcoRI and XhoI to give a DNA fragment of approximately 1.5 kb, which wasthen ligated to an EcoRI- and XhoI-digested DNA fragment (approximately9.2 kb) of plasmid pGAll (Appl. Environ. Microbiol., 63, 1362-1366(1997)) to thereby obtain plasmid pCAS-TRa2.

Example 11 Confirmation of Activity in Yeast Cells Expressing TRa2Protein on Their Surface

The plasmid pCAS-TRa2 was used to transform yeast S. cerevisiae strainEH1315. The resulting transformed strain was designated as strainTRa2-2. A loopful of the strain TRa2-2 or a control strain (strain C-2)was inoculated into 10 ml YPD liquid medium and cultured at 30° C. for 2days. The control strain was prepared by transforming yeast S.cerevisiae strain EH1315 with plasmid pCAS1 which had been designed tohave a multicloning site inserted into plasmid pGAll between thesecretion signal of the glucoamylase gene and a gene encoding the3′-half of α-agglutinin. The cells were collected by centrifugation,washed with PBS and then suspended in 1 ml PBS. This suspension was usedto perform sugar degradation reaction and glycosyltransferase reactionin the same manner as used in Examples 6 and 7, followed by analysis ofthe reaction products. The results confirmed that the strain TRa2-2 hadnot only dextrinase activity, but also glycosylation activity oncatechin using dextrin as a glycosyl donor, whereas the strain C-2 wasfree from these activities. Namely, the strain TRa2-2 was shown todisplay TRa2 in an active form on the yeast surface.

Example 12 Obtaining of TRa2 Homologs from Various Strains of the GenusTrichoderma

Trichoderma viride strain IFO31137, Trichoderma viride strain SAM1427,Trichoderma viride strain IF031327, Trichoderma viride strain IFO30498,Trichoderma viride strain IF05720, Trichoderma reesei strain IFO31329,Trichoderma reesei strain IFO31328, Trichoderma reesei strain IFO31326,Trichoderma saturnisporum strain IAM12535, Trichoderma ghanense strainIAM13109, Trichoderma koningli strain IAM12534, Trichoderma hamatumstrain IAM12505, Trichoderma harzianum strain IFO31292 and Trichodermapolysporum strain SAM357 were each inoculated into 10 ml liquid mediumcontaining 1% yeast extract (Difco), 1% polypeptone (NihonPharmaceutical Co., Ltd., Japan) and 2% dextrin (Nacalai Tesque, Inc.,Japan), followed by shaking culture at 30° C. for 4 days. The cells werecollected by filter filtration, and genomic DNA was extracted for eachstrain with a DNeasy plant mini Kit (QIAGEN). This genomic DNA (50 ng)was used as a template to perform PCR reaction with primers AMY-12f andAMY-15r. Namely, PCR was accomplished by using ExTaq (Takara Bio Inc.,Japan) under the following conditions: 94° C. for 2 minutes, (94° C. for1 minute, 50° C. for 1 minute, 72° C. for 1 minute)×30 cycles, and 72°C. for 10 minutes. The PCR products were analyzed by agarose gelelectrophoresis, confirming a fragment of approximately 0.6 kbp for eachstrain. These DNA fragments were each excised from the agarose gel andpurified with a GFX PCR DNA and Gel Band Purification Kit (AmershamBiosciences). Each DNA was cloned with a TOPO-TA cloning kit(Invitrogen) and analyzed for its nucleotide sequence using an ABI 3100Avant (Applied Biosystems). As a result, the sequences of SEQ ID NOs: 11to 24 were obtained. Their alignment results are shown in FIG. 8. Theidentity between the TRa2 gene and regions corresponding to thesesequences or between these sequences was found to be 63% to 99.5%.

On the basis of these sequences, it is possible to obtain TRa2 homologshaving the same activity as TRa2 when repeating the same procedure asused for obtaining the TRa2 gene of T. viride IAM5141 and itsfull-length cDNA.

Example 13 Analysis of Substrate Specificity of TRa2

The strain TRa2-1 was cultured overnight at 30° C. with shaking in 10 mlYPD medium. After reaching the resting phase, the culture solution wasinoculated into the same medium (2% (v/v)) and cultured with shaking at30° C. for 3 days. After culturing, the supernatant was collected bycentrifugation and concentrated 5-fold to give a crude enzyme solutionof TRa2. The reaction was performed at 45° C. for 24 hr in 100 μl enzymereaction solution containing 0.5 mM or 10 mM glycosyl acceptor compound((+)-catechin, (−)-epigallocatechin-3-O-gallate, esculetin, naringenin,quercetin, daidzein, genistein or kaempferol), 10 mg dextrin, 100 mMacetate buffer (pH 5.2) and the crude enzyme solution, followed by HPLCanalysis. The results obtained are shown in FIG. 9.

The area ratio (%) between acceptor compound and glycoside product was10% for (+)-catechin, 17.7% for (−)-epigallocatechin-3-O-gallate, 3.5%for esculetin, 4.4% for naringenin, 9.4% for quercetin, 10.7% fordaidzein, 6.8% for genistein, and 3.1% for kaempferol.

Example 14 Characterization of TRa2

Expression of His-tagged TRa2 protein (TRa2-His): Construction ofTRa2-His expression plasmid and obtaining of transformed yeast

To express a C-terminally His-tagged TRa2 protein in yeast cells, thefollowing primer was designed.

TRa2HisXhoI-r2: (SEQ ID NO: 28)Gctcgagttagtggtggtggtggtggtgtgaagacagcagcaa

The plasmid pCRTRa2-cDNA was used as a template to perform PCR reactionwith primers TraEcoRI-f2 and TRa2HisXhoI-r2. PCR was accomplished byusing Ex Taq (Takara Bio Inc., Japan) under the following conditions:94° C. for 2 minutes, (94° C. for 1 minute, 58° C. for 1 minute, 72° C.for 2 minutes) x 25 cycles, and 72° C. for 10 minutes. The resulting PCRproducts were analyzed by agarose gel electrophoresis, confirming a DNAfragment of approximately 1.5 kb. This DNA fragment was excised from theagarose gel and purified by GFX. The resulting DNA fragment was clonedwith a TOPO-TA cloning kit (Invitrogen), confirmed for its nucleotidesequence, and designated as plasmid pCRTRa2-cDNA-His. pCRTRa2-cDNA-Hiswas digested with EcoRI and XhoI to give a DNA fragment of approximately1.5 kb, which was then ligated to an EcoRI- and SalI-digested fragmentof plasmid pYE22m using ligation high (Toyobo Co., Ltd., Japan) tothereby obtain plasmid pYE-TRa2-His. The plasmid pYE-TRa2-His was usedto transform yeast S. cerevisiae strain EH1315. The resultingtransformed strain was designated as strain TRa2-3.

Culturing

The strain TRa2-3 was cultured in 20 ml SD(-Trp) at 30° C. for 16 hr.The pre-cultured solution was inoculated into 1 L of SD(-Trp) +100 mMKH₂PO₄-KOH (pH 6.0) and cultured at 30° C. for 3 days, followed bycentrifugation to collect the culture supernatant.

Purification

The culture supernatant was applied onto a Ni²⁺-chelated ChelatingSepharose Fast Flow (5 ml, Pharmacia Biotech) column equilibrated withBuffer Si [20 mM NaH₂PO₄-NaOH (pH 7.4), 10 mM imidazole, 0.5 M NaCl, 15mM 2-mercaptoethanol], followed by washing with the same buffer (40 ml).Subsequently, proteins bound to the column were eluted with Buffer El[20 mM NaH₂PO₄—NaOH (pH 7.4), 200 mM imidazole, 0.5 M NaCl, 15 mM2-mercaptoethanol]. Active fractions were collected, and then desaltedand concentrated using a VIVASPIN (30,000 MWCO, VIVASCIENCE).

Subsequently, the enzyme solution was applied (1.5 ml/min) onto aResource Q (1 ml, Pharmacia Biotech) column equilibrated with Buffer S2[20 mM KH₂PO₄-KOH (pH 7.4), 15 mM 2-mercaptoethanol, 0.1% CHAPS],followed by washing with the same buffer (10 ml). Subsequently, proteinsbound to the column were eluted with a 0-100% linear gradient of BufferE2 [20 mM KH₂PO₄-KOH (pH 7.4), 0.6 M NaCl, 15 mM 2-mercaptoethanol, 0.1%CHAPS] (60 ml). Active fractions were collected, and then desalted andconcentrated using a VIVASPIN (30,000 MWCO, VIVASCIENCE).

The same procedure was repeated again to perform Resource Q columnchromatography. Active fractions showing a single band on SDS-PAGE werecollected, and then desalted and concentrated using a VIVASPIN (30,000MWCO, VIVASCIENCE).

Measurement of Enzyme Activity: Glycosylation Activity

A reaction solution (100 μl, 10 mM catechin, 10 mg dextrin, 100 mMAcetate-NaOH (pH 5.3), enzyme solution) was stirred at 45° C. for 24 hr,followed by addition of 0.5% TFA (100 μl) to stop the reaction. Afterstopping the reaction, the sample was centrifuged to collect thesupernatant. The product was analyzed by HPLC under the conditions asshown below. HPLC conditions: Eluent A, 0.1% TFA; Eluent B, 90%acetonitrile, 0.08% TFA; analytical column, Develosil C30-UG-5 (4.6×150mm, NOMURA CHEMICAL); flow rate, 1 ml/min; separation mode, 0 min-5% B,20 min-50% B, 20.5 min-5% B, 25 min-5% B

Hydrolysis Activity

A reaction solution (100 μl, 5 mM maltotetraose, 20 mM KH₂PO₄-KOH (pH6.0), enzyme solution) was reacted at 35° C. for 15 minutes, followed bythermal treatment (100° C., 5 min) to stop the reaction. After stoppingthe reaction, the sample was centrifuged to collect the supernatant. Theproduct was analyzed by HPLC under the conditions as shown below. HPLCconditions: Eluent, 68% acetonitrile; analytical column, SUPELCOSILLC-NH₂ (5 μm, 4.6×250 mm, SUPELCO); flow rate, 1 ml/min; separationmode, isocratic

Temperature Stability Analysis:

The enzyme solution (±10 mM epigallocatechin gallate) was treated atdifferent temperatures ranging from 20° C. to 60° C. for 1 hr, and thencooled to 4° C. To the cooled samples, 10 mM epigallocatechin gallateand 10 mg dextrin were added and reacted (different temperatures, pH5.3, 24 hr). After addition of 0.5% TFA to stop the reaction, eachsample was analyzed by reversed-phase HPLC.

The results obtained are shown in FIG. 10 (left).

pH Stability Analysis:

To the enzyme. solution (±10 mM epigallocatechin gallate), a buffer ofdifferent pH (pH 4, 4.5, 5, Acetate-NaOH; pH 5.5, 6, 6.5, 7,NaH₂PO₄—NaOH; pH 8, 9, Tris-HCl) was added at a final concentration of167 mM. After treatment at 4° C. for 16 hr, Acetate-NaOH (pH 5.3) wasadded at a final concentration of 400 mM. To the treated samples, 10 mMepigallocatechin gallate and 10 mg dextrin were added and reacted (45°C., different pH, 24 hr). After addition of 0.5% TFA to stop thereaction, each sample was analyzed by reversed-phase HPLC. TheAcetate-NaOH concentration was 200 mM in reaction solution duringactivity measurement.

The results obtained are shown in FIG. 10 (right).

Analysis of Reaction Temperature Dependence:

The enzyme was added to 5 mM maltotetraose to perform hydrolysisreaction (different temperatures ranging from 20° C. to 60° C., pH 6.0,15 min). After stopping the reaction at 100° C. for 5 min, each samplewas analyzed by normal phase HPLC. The results obtained are shown inFIG. 11 (left).

In addition, the enzyme was added to 10 mM catechin and 10 mg dextrin toperform glycosyltransferase reaction (different temperatures rangingfrom 20° C. to 60° C., pH 5.3, 24 hr). After addition of TFA to stop thereaction, each sample was analyzed by reversed-phase HPLC. The resultsobtained are shown in FIG. 12 (left).

Analysis of Reaction pH Dependence:

The enzyme was added to 5 mM maltotetraose to perform hydrolysisreaction in a buffer of different pH (35° C., pH 4, 4.5, 5,Acetate-NaOH; pH 5.5, 6, 6.5, 7, NaH₂PO₄—NaOH; pH 8, 9, Tris-HCl, 15min). After stopping the reaction at 100° C. for 5 min, each sample wasanalyzed by normal phase HPLC. The results obtained are shown in FIG. 11(right).

In addition, the enzyme was added to 10 mM catechin and 10 mg dextrin toperform glycosyltransferase reaction (45° C., pH 4, 4.5, 5,Acetate-NaOH; pH 5.5, 6, 6.5, 7, NaH₂PO₄—NaOH; pH 8, 9, Tris-HCl, 24hr). After addition of TFA to stop the reaction, each sample wasanalyzed by reversed-phase HPLC. The results obtained are shown in FIG.12 (right).

Example 15 Characterization of Glycoside Heat Stability:

After 10 mM potassium phosphate buffer (pH 7.0, 30 μl) containing 100 μM(+)-catechin or 4′-O-α-D-glucopyranosyl-(+)-catechin was treated atdifferent temperatures ranging from 4° C. to 100° C. for 0 to 4 hours,each sample was transferred on ice and mixed with 0.1% TFA (60 μl),followed by HPLC analysis in the same manner as shown in Example 1. FIG.13 shows the % remaining of (+)-catechin or4′-O-α-D-glucopyranosyl-(+)-catechin when treated at differenttemperatures. The results indicated that4′-O-α-D-glucopyranosyl-(+)-catechin was more stable against heat thancatechin.

Solubility:

(+)-Catechin or 5-O-α-D-glucopyranosyl-(+)-catechin was added to waterat different concentrations ranging from 10 to 450 mg/ml and dissolvedby vigorous stirring, followed by centrifugation to remove precipitates.The supernatant was analyzed by HPLC to quantify the amounts of(+)-catechin and 5-O-α-D-glucopyranosyl-(+)-catechin. The resultsobtained are shown in FIG. 14. The results indicated that (+)-catechinwas substantially insoluble in water, whereas5-O-α-D-glucopyranosyl-(+)-catechin showed at least 40-fold or highersolubility.

INDUSTRIAL APPLICABILITY

The novel enzyme genes or modified variants thereof, transformantscarrying the same, and novel enzyme proteins or modified variantsthereof, which are obtained by the present invention, are useful inproducing glycosides of flavonoid compounds or in producing foods, drugsand/or cosmetics based on such glycosides.

1. An isolated protein comprising: (a) the amino acid sequence shown inSEQ ID NO: 10; (b) the amino acid sequence shown in SEQ ID NO: 10,except 1 to 9 amino acids are substituted, deleted, inserted, and/oradded, and which has glycosylation activity on a flavonoid compound; or(c) an amino acid sequence having at least 90% amino acid sequenceidentity to the amino acid sequence shown in SEQ ID NO: 10, and whichhas glycosylation activity on a flavonoid compound.
 2. An isolatedprotein comprising: (d) the amino acid sequence shown in SEQ ID NO: 26;(e) the amino acid sequence shown in SEQ ID NO: 26, except 1 to 9 aminoacids are substituted, deleted, inserted, and/or added, and which hasglycosylation activity on a flavonoid compound; or (f) an amino acidsequence having at least 90% amino acid sequence identity to the aminoacid sequence shown in SEQ ID NO: 26, and which has glycosylationactivity on a flavonoid compound.
 3. The protein according to claim 1,which is immobilized on a resin.
 4. The protein according to claim 2,which is immobilized on a resin.
 5. An isolated protein encoded by apolynucleotide comprising the nucleotide sequence of SEQ ID NO: 14 andhaving glycosylation activity on a flavonoid compound.
 6. The proteinaccording to claim 5, which is immobilized on a resin.